Establishment of a multiplex polymerase chain reaction assay for the detection of marine harmful algae

Various harmful microalgae coexist in the marine environment, seriously affecting the survival of aquatic animals, the sustainable development of fisheries, and pose a great threat to the safety of aquatic food. Consequently, monitoring these harmful microalgae in the marine aquaculture environment is an urgent issue that needs to be addressed. This paper focused on developing a multiplex polymerase chain reaction (MPCR) assay for the simultaneous detection of representative harmful microalgae commonly distributed in the aquaculture environment along the Chinese coast, including Karenia mikimotoi, Alexandrium tamarense, Chattonella marina, Prorocentrum minimum and Heterosigma akashiwo. First, the internal transcribed spacer and large subunit rDNA of target algal species were selected as target for the design of MPCR primers, the specificity of which was subsequently verified. Next, the MPCR system was established and the reaction conditions were optimized, including primer concentration, dNTP concentration, Mg2+ concentration, the concentration of Taq DNA polymerase, and annealing temperature. Specificity test showed that the established MPCR did not cross-react with all of the control algal species and thus can be used for specific detection of target algal species. The sensitivity test showed that the established MPCR could still detect all of the target algae when the concentration of genomic DNA was as low as 1 ng mu L-1. Moreover, the MPCR could still detect K. mikimotoi when the concentration of genomic DNA was as low as 1 pg mu L-1. The stability test indicated that the performance of the developed MPCR was not affected by the interfering algal species. In addition, the practicality of the established MPCR assay was evaluated using spiked water samples. The tests with the spiked samples indicated that the detection limit of MPCR for the all of the target algal species was approximately 4 cells mL-1, which can meet the requirement for the warning of HABs caused by them. In conclusion, the MPCR assay established here is characterized with strong specificity and good stability and is expected to be promising for daily monitoring of harmful microalgae in the aquaculture environment as an important alternative to traditional morphology-based methods.